There remains a compelling need for the development of treatments for unresectable melanoma. Agents that stimulate the innate immune response could provide advantages for cell-based therapies. However, there are conflicting reports concerning whether toll-like receptor (TLR) signaling controls tumor growth. The objective of this study was to evaluate the effect of intralesional administration of a TLR7 agonist in melanoma therapy. B16cOVA melanoma was implanted to TLR7 mice to evaluate the roles of stromal TLR7 on melanoma growth. To capitalize on the potential deleterious effects of TLR7 stimulation on the tumor growth, we injected melanoma tumor nodules with a newly developed and potent TLR7 agonist. B16 melanoma nodules expanded more rapidly in TLR7-deficient and MyD88 mice compared with TLR9 and wild type mice. Repeated injections with low doses of unconjugated TLR7 agonist were more effective at attenuating nodule size than a single high dose injection. To improve the efficacy we conjugated the agonist to phospholipid or phospholipids-polyethylene glycol, which retained TLR7 specificity. The phospholipid conjugate was indeed more effective in reducing lesion size. Furthermore, intralesional administration of the phospholipid TLR7 agonist conjugate enhanced the antimelanoma effects of systemic treatment with interleukin (IL)-2 and prolonged the survival of mice compared with IL-2 alone. Our study showed that: (1) TLR7/MyD88 signaling in the stroma is involved in melanoma growth; and (2) intralesional administration of a TLR7 agonist reduces the growth of melanoma nodules and enhances the antimelanoma effects of IL-2.